The aim of this project is to characterize the means by which target cells resist lysis by cytotoxic T lymphocytes (CTLs) and their toxic factors. CTLs kill targets by two pathways: a neurotic pathway involving formation of pores by the cytolysin, perforin, and an apoptotic pathway involving ligation of Fas molecules on target surfaces. CTLs, themselves, are highly resistant to lysis by other CTLs or by perforin or isolated secretory granules. Their membrane resists formation of perforin channels. This enables CTLs to survive attacks on target cells. Other cells, including CD4+ Th1 clones, enhance their survivability by removing perforin lesions through endocytosis and restoration of transmembrane potentials. Other factors also modulate sensitivity of target cells to CTLs and to perforin; these include position in the cell cycle and exposure to heat stress. Specific Aim 1. One aim is to identify the genes, and their products, expressed by CTLs that protect them from perforin. Though closely related to CTLs, murine Th1 clones succumb to perforin when depleted of ATP while CTLs do not. To enrich for genes responsible for the resistance of CTLs, we will utilize a subtracted cDNA library of CTL genes depleted of genes expressed by a Th1 clone. Genes will be identify by their ability to confer resistance to a perforin-sensitive lymphoma line. Also, components of cytotoxic granules are deposited in CTL surfaces upon secretion and may modify the membrane. Thus, we will fractionate the granules and evaluate the effect of fractions on the susceptibility of cells to perforin damage. Specific Aim 2. Another aim is to determine how changes in the cell cycle and exposure to heat stress alter susceptibility of target cells to T cell mediate cytotoxicity. Cells in late G1 are less susceptible to perforin than those at other stages of the cell cycle. We will determine whether their resistance is due to changes in ability to bind perforin or ability to repair their membranes. We will also examine how heat stress alters the susceptibility of target cells to CTLs and the role of the inducible isoform of heat-shock protein 70, i-hsp70, in preventing recognition of A20 lymphoma by cognate CTLs.